The differentiation of eukaryotic cells requires a hierarchy of regulatory mechanisms that involve a cascade of alteration in the transcription of specific genes. For example, specific gene expression (e.g., immunoglobulin genes) in pre-B and mature B cells is activated by a family of bHLH (basic helix-loop-helix) transcription factors. In progenitor B cells, however, the function of these bHLH transcription factors is suppressed by a group of inhibitory Id proteins whose expression diminishes only when the progenitor B cells are triggered to differentiate. The primary objective of my proposal is to understand the hierarchy of transcriptional events that dictates B cell development, through studying the regulation of the Id genes and the consequences of their dysregulation during B cell development. The findings of these studies may also shed light on mechanisms of differentiation operating in other cell types. Because the Id proteins are thought to be inhibitors of cell differentiation, knowledge about the regulation of the Id genes in B cell development may help to understand certain developmental aberrations that result in immunodeficiency, autoimmunity and malignancy. This research proposal includes the following two specific aims: (1) Investigation of the molecular mechanism of transcriptional regulation of the Id1 gene, including the identification of cis-acting elements and trans-acting factors for the down-regulation of the Id1 gene during B cell differentiation. The flanking sequences of the Id1 gene will be analyzed by means of deletion and point mutations and assayed for enhancer and silencer activities in B cell lines. The proteins binding to the identified cis-acting elements will be characterized by in vitro footprinting, in vivo genomic footprinting and electrophoretic mobility shift assays. (2) Determination of the effects of altered Id gene expression on the transcription of B cell-specific genes and the rearrangement of the immunoglobulin genes. The Id cDNAs will be over- expressed in pre-B and mature B cells using various expression vectors and Id expression will be reduced in pro-B cells by antisense oligonucleotides or antisense RNA. We will also generate transgenic mice which carry Id genes driven by constitutive B cell-specific promoters such as that of the mb-1 gene. We will determine where dysregulated Id expression in these transgenic mice can block B cell differentiation by monitoring the development of mature B cells using FACS analyses of bone marrow and spleen, and whether such blockage of differentiation causes certain malignancy in the immune system using hematopathological analyses of blood and bone marrow.